IN VIVO AMPLIFICATION

Question 1

Amplifying dna

Answer 1

making lots of copies of dna from the fragment made from recombinant technology so we have sufficient dna to work with.

Question 2

in vivo cloning

Answer 2

• copies of dna fragments are made inside the body of a living organism.

Question 3

modifying dna fragment

Answer 3

• restriction endo nuclease are used to cut out the dna fragment
• these enzymes cut at recognition sites leaving sticky ends
• a promoter region and a terminator region must be added.

Question 4

promoter region

Answer 4

• promoter region must be added at the start of the dna fragment.
• this is a sequence of dna which is a binding site for rna polymerase to initiate transcription.

Question 5

terminator region

Answer 5

• terminator gene is then added into the end of the dna fragment.
• this is added to detatch rna polymerase and stop transcription so only one gene is copied into mrna at a time.

Question 6

vector

Answer 6

something that is used to transfer isolated dna fragment into a host cell.
-plasmids( circular loops of dna separated from the main bacterial genome ) are the most commonly used vectors.

Question 7

inserting a dna into a vector

Answer 7

• plasmid is cut open using the same restriction endonuclease enzyme.
• this creates the same sticky ends
• now the sticky ends of dna are complementary to sticky ends of a plasmid.
• using the same enzyme is necessary so that the same sticky ends are created at both the dna fragment and plasmid so that they are complementary to each other.
• the vector dna and dna fragment are mixed together with an enzyme dna ligase which joins the sticky end of vector dna and sticky end of dna fragment which is called ligation.
• recombinant dna is fromed

Question 8

transformation ( bacteriophage vector)

Answer 8

• if a bacteriophage vector is used, then bacteriophage will infect the host bacterium by injecting its dna into the cell.
• host cells are said to be transformed if they take up the vectors containing the gene of interest.

Question 8

ligation and role of ligase

Answer 8

the enzyme dna ligase sticks or anneals the dna fragment and the vector dna together which is what we call ligation.
- ligase catalyses the condensation reaction to form phospho diester bonds between nucleotides.

Question 8

recombinant dna

Answer 8

• new combination of bases in dna ( vector dna + dna fragment)

Question 8

issues in identifying transformed cell

Answer 8

1. recombinant dna doesnt enter the cell
2. plasmid rejoins before the dna fragment is entered.
3. dna fragment sticks to itself rather than sticking to plasmid

Question 9

transformation ( plasmid vector)

Answer 9

the vector ( plasmid with re combinant dna) then needs to be transferred into host cell where the gene will be expressed to create a protein.
- if a plasmid vector is used, firstly the cell of host cell needs to be made more permeable.
- to make the cells more permeable, host cells are mixed with calcium ions and heat shocked to increase the permeability which enables vector to enter the cytoplasm of host cells.

Question 9

identifying transformed cells

Answer 9

• not all host cells take up vector and its dna so its important to identify which cells have been transformed.

Question 10

marker genes

Answer 10

genes on the plasmid used to identify which bacteria succesfully took up the recombinant plasmid.

Question 10

three different marker genes

Answer 10

1. antibiotic resistance genes
2. genes coding for fluorescent proteins
3. genes coding for enzymes.

Question 10

genes coding for fluorescent proteins

Answer 10

• jellyfish contain a gene that code for gfp ( green fluorescent protein)
• gfp is inserted into a bacterial plasmid.
• dna fragment is inserted in the middle of gfp which disrupts and prevent gfp production.
• only the non glowing ones contain the recombinant plasmids.

Question 11

enzyme marker

Answer 11

• enzyme lactase can turn a certain substance from colourless to blue.
• gene for this enzyme is inserted in a plasmid
• dna fragment is inserted in the middle of the gene to disrupt it
• bacteria grown on agar plate with colourless substance
• colonies which cannot tuen colourless substance into blue contain the recombinant plasmids.

Question 12

fermenter

Answer 12

• the host cells containing recombinant plasmids are grown in a fermenter .
• the large cloned population can produce protein which is coded by the inserted dna fragment.
• eg bacteria producing insulin from inserted insulin gene.